A remarkable property of RNA interference (RNAi) in C. elegans is its association with intercellular RNA transport pathways. This linkage mobilizes dsRNA-silencing signals and enables silencing to spread from the site of initiation throughout the animal and to the progeny. This phenomenon, known as systemic RNAi, is a conserved process among many multicellular organisms. Through genetic analysis, we have isolated systemic RNAi defective mutants (sid) and have identified the corresponding proteins (SID). SID-1 is a widely conserved dsRNA channel that selectively and specifically transports dsRNA into cells and is essential for systemic RNAi. A mammalian SID-1 homolog is critical for cytoplasmic delivery of modified siRNAs, suggesting that dsRNA transport is a conserved function for this family of channel proteins. SID-2 is a putative dsRNA receptor that is expressed and localized exclusively to the luminal membrane of the intestine. SID-2 transports ingested dsRNA across the intestinal epithelium into the animal to trigger RNAi. This process of sequence-specific gene silencing in response to environmentally-encountered dsRNA, known as environmental RNAi, is widespread throughout nature, including in mammals. The development of RNAi-based drugs enables targeting of previously """"""""undruggable"""""""" disease related genes and has exciting therapeutic potential. However, the efficacy and safety of RNAi as a gene-silencing therapy requires understanding how therapeutic dsRNAs enter cells to gain access to the silencing machinery. In addition, we must rationally determine how to modify these dsRNA molecules for more efficient delivery and targeting to select tissues and cells. Our analysis of dsRNA transport through the SID-1 channel indicates that even minor modifications, such as removing an oxygen atom from the ribose sugar, can block dsRNA transport. Thus, understanding the regulation and function of these proteins in the experimentally tractable nematode C. elegans will provide valuable insights for the advancement of RNAi-based drugs as a novel class of therapeutic agents in humans. The long-term objective of the proposed research is to understand the physiological importance and mechanism of intercellular RNA transport in animals. Towards this end, the specific aims of this proposal are: 1) To characterize the specificity and regulation of the SID-1 dsRNA channel; 2) To characterize ingested dsRNA uptake mechanism in environmental RNAi; 3) To characterize extracellular dsRNA transport pathways;and 4) To isolate and characterize endogenous extracellular RNAs in C. elegans.
These aims address, through a combination of genetic, biochemical and biophysical approaches, questions at the leading edge of the recently discovered field of intercellular RNA transport and further explore the possibility that extracellular RNA molecules underlie a novel means of signaling in multicellular organisms. Remarkably, this process, which was unknown 10 years ago, now has immediate clinical relevance.

Public Health Relevance

The specificity and potency of dsRNA-based gene silencing lends tremendous hope for the treatment of a wide range of human diseases including cancer. Although clinical trials for several RNAi-based drugs are already underway, the mechanisms underlying dsRNA transport and uptake are poorly understood. Understanding how dsRNA crosses cell membranes to trigger RNAi will lead to improvements in targeting and delivery efficiency, and will limit potential deleterious side effects of this exciting therapeutic strategy.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Bender, Michael T
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Harvard University
Schools of Arts and Sciences
United States
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Minkina, Olga; Hunter, Craig P (2018) Intergenerational Transmission of Gene Regulatory Information in Caenorhabditis elegans. Trends Genet 34:54-64
Whangbo, Jennifer S; Weisman, Alexandra S; Chae, Jeiwook et al. (2017) SID-1 Domains Important for dsRNA Import in Caenorhabditis elegans. G3 (Bethesda) 7:3887-3899
Minkina, Olga; Hunter, Craig P (2017) Stable Heritable Germline Silencing Directs Somatic Silencing at an Endogenous Locus. Mol Cell 65:659-670.e5
Wang, Eddie; Hunter, Craig P (2017) SID-1 Functions in Multiple Roles To Support Parental RNAi in Caenorhabditis elegans. Genetics 207:547-557
Shiu, Philip K; Hunter, Craig P (2017) Early Developmental Exposure to dsRNA Is Critical for Initiating Efficient Nuclear RNAi in C. elegans. Cell Rep 18:2969-2978
Nguyen, Tan A; Smith, Blake R C; Tate, Michelle D et al. (2017) SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition. Immunity 47:498-509.e6
Yang, Huan; Vallandingham, Jim; Shiu, Philip et al. (2014) The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans. Curr Biol 24:832-8
Schott, Daniel; Yanai, Itai; Hunter, Craig P (2014) Natural RNA interference directs a heritable response to the environment. Sci Rep 4:7387
Shiu, Philip K; Zhuang, Jimmy J; Hunter, Craig P (2014) Assays for direct and indirect effects of C. elegans endo-siRNAs. Methods Mol Biol 1173:71-87
Zhuang, Jimmy J; Banse, Stephen A; Hunter, Craig P (2013) The nuclear argonaute NRDE-3 contributes to transitive RNAi in Caenorhabditis elegans. Genetics 194:117-31

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